Airway alkalinization as therapy for airway diseases

ABSTRACT

The present invention relates to a method of treating asthma by raising the pH of the airways of an individual. The effect can be mediated directly by administering a pharmaceutically acceptable basic solution or alternatively, the effect can be mediated by enhancing the activity of glutaminase.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of copending application Ser.No. 11/133,663, filed May 20) 2005, which is a divisional of applicationSer. No. 10/181,840, filed Jul. 8, 2002, which was filed as a 371 ofinternational PCT/US01/01062 having a PCT filing date of Jan. 12, 2001,which international application claimed the benefit of U.S. ProvisionalApplication No. 60/176,388, filed Jan. 14, 2000. All of these relatedapplications are incorporated herein by reference in their entirety.

US GOVERNMENT RIGHTS

This invention was made with United States Government support underGrant No. RO8L59337-01, awarded by The National Institutes of Health.The United States Government has certain rights in the invention.

FIELD OF THE INVENTION

The present invention is directed to methods and compositions fortreating asthma and other airway diseases. In particular, the presentinvention is directed to compositions and methods that raise the pH ofthe bronchial airways to alleviate the symptoms associated with asthmaand other airway diseases.

BACKGROUND OF THE INVENTION

Concentrations of reactive oxygen and nitrogen species are high in theexpired air, condensed breath and bronchoalveolar lavage fluid ofpatients with asthma. The bioactivities of many of these compounds inthe asthmatic airway are critically dependent on pH in vitro. Forexample, protonation of nitrite (NO₂ ⁻) to form nitrous acid (HNO₂;pKa˜3.3) and protonation of peroxynitrite (ONOO⁻) to form peroxynitrousacid (ONOOH; pKa˜6.3) converts relatively inert salts into highlycytotoxic species. These acids may cause substantial injury to theairway epithelium, largely through the generation of free radicalsaccording to reactions (1) and (2).

2H⁺+2NO₂ ⁻→2HNO₂→H₂O+.NO₂+.NO  (1)

H⁺+ONOO⁻→ONOOH→.OH+.NO₂  (2)

Because of the pH-dependent cytotoxicity of chemical species present inhigh concentrations in the asthmatic airway, the pH of expired water insubjects with acute asthma was studied. This water was condensed duringtidal breathing. Samples were filtered during collection (0.3 μm) andwere deareated with argon to eliminate artifact caused by variablecarbon dioxide tensions. pH values were highly reproducible andunrelated to salivary pH, to nebulizer therapy or to gastroesophagealreflux history.

The mean pH in subjects with acute asthma was over two log orders lowerthan in controls (7.65±0.20 vs. 5.23±0.21; n=19 and 22, respectively,p<0.001; representing in excess of 100 fold increase in proton/hydronium(H₃O⁺) concentration). These findings are consistent with evidence forlow nasal epithelial pH in patients with rhinitis and withbronchoalveolar lavage acidification in ovalbumin-sensitizedguinea-pigs. Furthermore, endogenous administration of an acid aerosolto human and animal airways is an effective and reproducible method forcreating asthma symptoms. Specifically, an acid aerosol treatment causesairway smooth muscle constriction and airway epithelial injury, classicfeatures of an acute attack of asthma. Moreover, low airway pH causesthe death of inflammatory cells lining the airway with release of toxicmediators that perpetuate inflammation and bronchoconstriction, andlikely contributes to elevated levels of nitric oxide found in theexhaled breath of asthmatic patients.

Increased serum and airway ECP levels and increased nitric oxide (NO)production/nitrogen oxide (NO_(x)) toxicity have both been consideredmarkers for worsening asthmatic airway inflammation. These effects ofairway acidification thus provide not only a theoretical modelexplaining cytotoxicity and airflow obstruction in asthma exacerbations,but also describe the specific findings observed during asthmaexacerbations in general. Of note, several respiratory epithelial cellfunctions, such as ciliary motility, are also impaired at low pH, andairway mucous production is increased. It has not previously beensuspected that these elements of asthma pathophysiology could bestimulated by endogenous airway acidification.

Endogenous acidification of airway lining fluid may be beneficial duringcertain infections as an innate host defense mechanism. However, inpatients with asthma, this process could be expected to aggravate airwayinflammation. Our evidence suggests two important mechanisms by whichthis acidification selectively affects asthmatic subjects. First,acidification may selectively injure the asthmatic airway throughtoxification of reactive chemical species. Second, necrosis of residentairway eosinophils caused by a fall in pH will lead release ofinflammatory mediators such as eosinophil cationic protein.

SUMMARY OF THE INVENTION

The present invention is based on the premise that endogenous airwayacidification, to the degree observed in breath condensates fromsubjects with acute asthma, is uniquely toxic to the allergic asthmaticairway. An important mechanism by which this acidification occursinvolves inhibition of airway epithelial glutaminase by Th1-derivedcytokines and by GSNO. Accordingly, based on this discovery, severalnovel therapeutic strategies for the prevention and treatment of asthmaexacerbations can be employed including 1) corticosteroid timing anddosing based on breath condensate pH and/or NH₄ ⁺ measurements; 2)inhibition of airway epithelial γ GT; 3) identification of novelinducers of glutaminase; 4) dietary or airway supplementation withglutamine during viral respiratory infections for patients with asthma;5) use of alkalinizing aerosols during asthma exacerbations; and/or 6)aerosolized use of inhibitors of other airway acidification mechanisms,such as of carbonic anhydrase or vacuolar (V) ATPase.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing ammonium concentrations are low in breathcondensate specimens from patients with acute asthma. Specimens werecollected from patients admitted to the hospital or seen in theemergency department with acute exacerbations of asthma (low pH) or fromhealthy volunteers. The median NH₄ ⁺ concentration in asthmatic subjects(37; range, 0-442) was lower than in controls (314; range, 14-220) byMann-Whitney rank-sum testing (p<0.001).

FIG. 2 is a graphic demonstration showing that inhalation ofphosphate-buffered saline decreases expired NO concentration in asthma.Nebulized treatment with 0.05 mL/kg 10 mM PBS at time 0 results in atime-dependent decrease in expired NO concentration in acute asthma.

FIG. 3 is a graphic demonstration showing that breath condensate pHnormalizes during corticosteroid treatment for acute asthma. Serial pHmeasurements were made on five hospitalized patients with asthma andreceiving corticosteroid treatment. Geometric mean pH after treatment(7.5±0.23) was over two logs than at the initiation of treatment(5.3±0.38; p=0.001).

FIG. 4 is a graphic demonstration showing that experimental HRV 16infection is associated with increased upper airway ECP levels. Tennon-allergic, non-asthmatic subjects (-), 11 subjects with asthma andrelatively low IgE values (29.2-124 IU/mL) (

) and six subjects with high IgE values (371-820 IU/L) and asthma (

) were innoculated intranasally with HRV 16, 500 tissue cultureinfective dose 50% (TCID₅₀)/mL, 2.5 mL in each nostril twice at time 0.Nasal lavage ECP levels were measured sequentially for three weeks.

FIGS. 5A and 5B represent a graphic demonstration showing that infectionwith human rhinovirus causes a fall in pH and increased in expired NO insubjects with asthma who have an IgE>200 IU/mL. Asthmatic (FIG. 5A) andcontrol (FIG. 5B) subjects were innoculated with HRV 16 at time 0.Breath condensate and expired NO was measured at various time pointsafter innoculation. Maximal fall in pH and rise in expired NO occurred48 hours after innoculation in high-IgE asthma.

DETAILED DESCRIPTION OF THE INVENTION

In accordance with one embodiment the symptoms of asthma are alleviatedby administering a pharmaceutically acceptable basic solution, having apH of about 7.4 to about 10, to a patient in a form that contacts theairways of the patient. More particularly, the present invention can useany base, delivered by aerosol to the airway, for the purpose oftreating asthma. The goal will be to decrease the proton concentrationin the airway lining fluid from approximately 10 μM to approximately 100nM (10⁻⁵/10⁻⁷).

The protons (or H₃O⁺ ions depending on nomenclature) are buffered tovarying degrees in the airways lining fluid, and therefore a molarequivalent of base will not necessarily be sufficient to normalize pH.Instead, an adequate amount of base needs to be provided to the lowerairway to react with the protons as they are freed from the airwaylining fluid buffer system. This may range from approximately 10⁻⁵ Eq/Lin an essentially unbuffered lower airway fluid to 10⁻⁴ in a more highlybuffered system. Given that this airway lining fluid is ordinarilyapproximately 1 ml/kg body weight, this will be approximately 10⁻⁸ to10⁻⁷ mEq/kg. However, only approximately 10% of an aerosol iseffectively delivered to the lower airway. Therefore, the appropriatedose of base would be approximately 0.1 to 1.0 μMole/kg.

The base used could be an endogenous stable base such as phosphate or anamine, or a synthetic, non-toxic base. In one embodiment the base usedis nebulized sodium phosphate monobasic or sodium acetate, or otherphysiologically acceptable salt of those compounds. The base can beadministered alone or in conjunction with glutamine (i.e. administeredas a mixture of the base with glutamine or the two components areadministered separately but within a short time frame, for example, thesecond being administered within three minutes of the first).

This therapy would be delivered early in the course of an acute asthmaexacerbation, when airway pH begins to fall. It would thus prevent theneed to use systemic steroids to treat or prophylax against an asthmaexacerbation. It could be used in conjunction with measurement of airwaypH by condensation technique. That is, the use of base could be judged,and the dose of base titrated, according to the endogenous airway pH asmeasured by condensation technique using techniques known to thoseskilled in the art. This would allow for precise and “natural,” nontoxictherapy for asthma exacerbations.

The idea of using natural compounds and adjusting therapy according toendogenous pathologic response is likely to have tremendous appeal topatients adverse to using pharmacologic agents for asthma management,and is likely to not have the toxicities associated with corticosteroidsanti-inflammatory therapy and μ-agonist smooth muscle relaxants.

In another embodiment of the present invention, alkaline buffer systemscan be delivered to the airway. Such systems would include, but not belimited to, combinations of ammonia (NH₃) and conjugate acids of NH₃such as ammonium acetate, ammonium chloride, or ammonium bicarbonate, ina system with concentrations of base/conjugate acid and added salts(such as sodium chloride) such that the pH, pK_(a), buffer capacity, andionic strength of the fluid are best suited to use for treatment ofvarious levels of acidity in the asthmatic airway. Additionally, otherbasic buffer systems could be used, such as, but not limited to, glycine(aminoacetic acid), pK_(a)2=9.78), bicine(N,N-Bis(2-hydroxyethyl)glycine (pK_(a)=8.46), tricene(N-[tris(hydroxymethyl)methyl] glycine pK_(a)=8.26), CAPS(3-(Cyclohexamino)-1-propanesulphonic acid (10.51), CAPSO(3-(Cyclohexamino)-2-hydroxypropanesulphonic acid (pK_(a)=9.71),2-(Cyclohexamino)-ethenesulphonic acid (pK_(a)=9.41), to name a few. Thechoice of buffer system could also be individualized for patientsdepending on results of non-invasive measurements of airway redoxchemistry (based on airway vapor condensate assays).

In an alternative embodiment of the present invention, pharmacologicagents are administered to an asthmatic that prevent the production ofacid in the airway, and/or enhance the production of base in the airwayendogenously, as a treatment for asthma. We have also discovered thatmean breath condensate concentrations of acetic acid in subjects withacute asthma (9.5 mg/L; n=9) are over 4 times higher than those incontrols (1.76 mg/L; n=18), in part explaining the lower pH in theasthmatic samples. Decreasing acetic acid production would therefore bean effective treatment for asthma For example, a patient can be treatedwith a nebulized formulation or treated systemically with a compoundthat inhibits aldehyde dehydrogenase (such as Disulfiram), to preventformation of acetic acid in the lung. Alternatively, or in addition toother therapies, a patient can be treated with a nebulized formulationor treated systemically with antifungal agents such as fluconazole,which kills yeast that may ferment sugars and provide substrate foracetic acid formation in the lung.

The airway epithelial surface can maintain a constant pH at differinglevels of pCO₂, and this homeostasis has been attributed to a variety ofepithelial cell functions including carbonic anhydrase activity,basolateral Na⁺/H⁺ exchange and lumenal HCO₃ ⁻ exchange through Cl⁻channels. However, applicants have discovered an additional mechanism bywhich airway pH may be regulated, involving the production of NH₃ byglutaminase in human airway epithelial cells. Robust mRNA and proteinexpression for the human kidney (hKGA) and human C (hGAC) isoforms ofglutaminase are evident in human lower airway epithelial cellpopulations. Therefore, one aspect of the present invention is based onthe principle that glutaminase-derived NH₃ buffers acid in the airway(as it does in the kidney) by forming airway lumenal NH₄ ⁺. In additionapplicants have shown that NH₄ ⁺ concentrations are low in the expiredair condensates of patients with acute asthma who have low pH (median:range=37; 0442 microM; n=23) compared to controls (314: 14-1220 microM;n=16; p<0.001). Thus in accordance with one embodiment, asthma can betreated by administering aerosolized glutamine.

Glutaminase activity increases in the renal tubular epithelium tocompensate for epithelial acidification. Expression is also stronglyupregulated by corticosteroids and inhibited by certain inflammatorycytokines. Compensatory stabilization of glutaminase mRNA in response toan environmental acid load has recently been demonstrated to involvebinding of a cytoplasmic protein to an eight-base AU repeal. The onlyknown promoter for the glutaminase gene is dexamethasone-responsive,consistent with observations that corticosteroid treatment increasesairway pH in asthmatic subjects suffering from an acute exacerbation(see FIG. 5). Moreover, glutaminase expression is consistently inhibitedby interferon γ (IFN γ) in the kidney and airway.

In accordance with one embodiment of the present invention a method fortreating asthma comprises administering a pharmaceutically acceptablecompound that either directly or indirectly raises the pH of thebronchial airways. As used herein the term treating includes alleviatingthe symptoms associated with a specific disorder or condition and/orpreventing or eliminating said symptoms. Examples would include, but notbe limited to, inhibition of airway V ATP'ases (which produce acid) andstimulation of airway epithelial glutaminase (which can produce base).The activity of glutaminase is inhibited by Th1 cytokines and isupregulated by corticosteroid therapy. Agents that inhibit airway acidproduction can be delivered either by aerosol or systemically in oralparenteral form. In one embodiment the therapy would also includeproviding a substrate to glutaminase (specifically glutamine) and otherenzymes capable of producing ammonia (a base produced endogenously inthe airway).

The present disclosure also encompasses the use of conventional agentssuch as Albuterol or Ipratroprium, currently used in day-to-day practiceof asthma management, at more alkaline pH. Existing formulations areadministered a an acidic pH:

Albuterol, pH 3 to 5

Levalbuterol HCl, pH 3.3-4.5

Ipratroprium bromide, pH 3-4

In accordance with one embodiment of the present invention an improvednebulizer solution is provided wherein the pH of existing formulationsare modified to have a pH of greater than 7, and in another embodiment apH of about 7.5 to about 9.5. This would involve alkalinization of themedicine immediately prior to use by mixing in a preformed diluent(phosphate buffer pretitrated to the acidity of the medicine, forexample) as opposed to normal saline. It also embodies the mixture ofthese conventional agents in an alkaline buffer system capable ofneutralizing airway acids. In one embodiment these standard compositionscan be combined with glutamine to supply a substrate for glutaminase andthus further alkalinate the bronchial airways.

In one embodiment, a kit is provided for treating asthma. The kitcomprises a conventional asthma compound, for example albuterol,levalbuterol HCl, ipratroprium bromide, or budesonide in solution, and abuffered diluent, wherein the diluent will effectively raise the pH ofthe solution to a basic pH. Suitable buffers are well known to thoseskilled in the art and a buffer can be selected that does not interferewith the activity of the conventional asthma agent.

In accordance with the present invention, asthmatic patients can betreated by lowering the pH of fluids that asthmatic contact during anordinary day. For example, the present invention also includes the useof alkaline humidification of the air for treatment and prevention ofasthma exacerbations. Furthermore, household water can be alkalinized(with such agents as soda ash) and used for the specific purpose oftreating asthma or airway diseases associated with an acidic airwaylining fluid. In one embodiment, part of the therapy for treating asthmaprophylactically comprises the use of environmental filters to scrubprotons (acid rains and acid fogs) that may aggravate endogenous airwayacidification during conditions of atmospheric pollution, thus worseningor predisposing to an asthma exacerbation.

This disclosure also encompasses the use of methods described in theabove paragraphs in conjunction with traditional methods of asthmamanagement. Further, it claims the use of normalization of airway pH asa measure of efficacy by which to titrate the use of conventional asthmatherapies, such as systemic and inhaled glucocorticoids. The use ofglucocorticoids for treatment of asthma is consistent with the fact thatthe only known promoter for the glutaminase gene is dexamethasoneresponsive, and that corticosteroid treatment increases airway pH inasthmatic subjects suffering from an acute exacerbation. Therefore theeffect of glucocorticoid treatment may be mediated through thestimulation of glutaminase expression. The dose of oral glucocorticoids(Prednisone, for example) a patient may take may be tapered ordiscontinued once the airway pH is normalized. This therapy could beused to decrease the level of intervention for patients with severeasthma who are chronically on relatively high doses of glucocorticoids.In this embodiment, daily or repeated daily use of an acrosolized basewould be used to decrease the dose of potentially more toxic compoundsrequired to keep the patient asymptomatic. One compound suitable for usein accordance with the present invention is aerosolized glutamine.

The methods described above can also be used to modify the pH of thenasal fluids, particularly in regards to treatment of symptoms andunderlying mechanisms of pathology for allergic rhinitis and common coldviruses such as rhinovirus and other airway based infections. Bothallergic rhinitis and common cold viruses likely decrease the pH ofnasal secretions, with various toxicities thereby resulting. Inparticular, viral infections, by stimulating a Th1 and/or CD8 lymphocyteresponse, may initiate the airway acidification that ultimatelycontributes to asthma symptoms, and thus modifying the pH of thepatients airways may alleviate symptoms associated with such infections.

Human Rhinovirus (HRV) infections are a leading cause of acute asthmaexacerbations in children over two, as well as in adults. The mechanismby which HRV selectively initiates expiratory airflow obstruction inpatients with asthma is not completely understood, however airway pHhomeostasis may play an important role. Acute asthma exacerbationscommonly follow HRV infections. Applicants have recently demonstratedthat breath condensate pH and NH₄ ⁺ levels fall dramatically and inparallel with a fall in condensate ammonium levels from a median of 351(range 152-1585) microM to 65 (4-713) microM p<0.05 in asthmaticsubjects experimentally inoculated with HRV strain 16 in human subjectsapproximately 72 hours following experimental HRV 16 infection (seeFIGS. 5A and 5B). Because this full in pH could lead to asthma symptomsin patients with eosinophilic airway inflammation and high levels ofNO_(x), this drop in pH likely contributes to lower airway symptoms inasthmatic patients who develop an upper respiratory infection.Rhinovirus infection could lead to inhibition of glutaminase and a fallin lung water pH by several mechanisms, including, 1) direct infectionof lower airway respiratory epithelial cells; 2) a cytokine-mediatedinflammatory response; and/or 3) a neural signaling pathway.

Direct infection of the lower airway with HRV during acute asthmaexacerbations has been postulated. However, the role of glutaminase indirect infection has not been described, and the direct effects of HRVinfection alone an airway epithelial cells (in the absence of an immuneresponse) are subtle. The role of glutaminase in HRV-exacerbatedasthmatic airway disease relates to its ability to modulate airway pH.Acidification of the airway is cytotoxic and contributes to asthmaticairway disease by 1) protonating nitrite (NO₂ ⁻) and peroxynitrite(ONOO⁻) to form membrane-destructive species such as hydroxyl (.OH) andnitrogen dioxide (.NO₂) as well as nitric oxide (.NO); 2) eosinophilnecrosis; 3) increased mucous secretion and 4) impaired ciliary beating.Airway acid is buffered by ammonia (NH₃) evolved from glutaminasederived ammonium (NH₄ ⁺) with basolateral loss of H⁺ in exchange forNa⁺.

An indirect effect of HRV infection involving inflammatory and/orneuronal mediators is also possible. In particular, IFN γ is a likelycandidate to mediate glutaminase inhibition because 1) Th1 cytokinesinhibit glutaminase activity in lower airway epithelial cells 2) IFN γinhibits glutaminase in other cell systems; and 3) IFN γ productionfollowing HRV infection in the setting of Th2-lymphocyte associatedchronic inflammation may contribute to asthmatic airway narrowing.Applicants have shown that 100 u/ml interferon gamma inhibitsglutaminase activity in cultured lung epithelial cells in excess of 65%at 24 and 48 hours (p<0.01, n=3 each). Though circulating T cells couldproduce IFN γ during rhinovirus infections, it seems more likely that itwould be secreted in the airway itself as a result of local recruitmentand activation of Th1 and/or CD8 cells.

Applicants have shown that a class of endogenous neuroregulatorymolecules known as S-nitrosothiols also inhibit glutaminase activity100% at 24 hours (p<0.01, n=3), probably through S-nitrosylation of acritical cysteine on the enzyme. In accordance with one embodiment, amethod of treating asthma comprises nebulizing or treating systemicallywith a compound that facilitates breakdown of S-Nitrosothiols, such asCuCl, Cu/Zn SOD, glutathione peroxidase, glutathione-dependentformaldehyde dehydrogenase or gamma glutamyl transpeptidase. Thepresence of S-nitrosothiols in the airways may be derived from airwayepithelial cells themselves during Th1-stimulated nitric oxide synthase2 upregulation, perhaps providing a cellular mechanism by which IFN γinhibits glutaminase. IFN γ's role in inhibiting glutaminase may offeryet another means of treating asthma that can be used alone or inconjunctions with the other methodologies of the present invention. Inone embodiment, an asthmatic patient can be treated with a nebulizedformulation or treated systemically with an antibody to interferongamma.

Additionally, the presence of S-nitrosothiols in the airways may arisefrom other inflammatory cells in the airway and/or from non-adrenergic,non-cholinergic nerves in the airway wall. Of note, though SNOs tend tobe catabolized rapidly in the airway lumen of asthmatic humans andovalbumin sensitized guinea pigs, a critical catabolic enzyme, γglutamyl transpeptidase (GGT), bioactivates the most common of theseSNOs, S-nitrosoglutathione (GSNO), forming the cell-permeable product,S-nitrosocyteinyl glycine (CGSNO). This pathway from GSNO to thebioactive S-nitrosylating agent CGSNO is relevant both in the nervoussystem and in the lung, and may be involved in signaling the inhibitionof glutaminase during HRV infections.

Taken together, these observations suggest that HRV causes lung water pHand NH₄ ⁺ levels to fall through inhibition of glutaminase, whetherthrough direct infection of the lower airway cells, throughlymphocyte-mediated pathways and/or through neural activation.Accordingly, methods that counteract the inhibitory effect onglutaminase, or raise airway pH may be effective in treating HRVinfections-induced asthma exacerbations.

In another embodiment of the present invention airway acidosis mediatedthrough protonation reactions involving reactive nitrogen and oxygenspecies may have antimicrobial effects. In particular nitriteacidification has been proposed as a mammalian host defense mechanism.The abundant NO₂ ⁻ of the airway is present as bacteriotoxic HNO₂ inrelevant quantities only when the pH is low. Mycobacterium tuberculosisproduces a gene product specifically protecting against the effects ofHNO₂. Some of the toxicity of HNO₂ occurs because of its reactivedecomposition to NO, which is known to inhibit mycobacterial growth.Additionally, hydrogen peroxide, elevated in the condensed breath ofasthmatic patients, acts synergistically with HNO₂ to kill gramnegative, mycobacterial and viral organisms. Applicants believe this tobe the reason that the human organism has adopted the mechanisms we havediscovered to acidify lung water. For example, as is shown herein, lungwater pH and ammonium level fall in response to HRV infection (see FIGS.5A and 5B). As such, restoration and/or augmentation of the acidifyingmechanisms of the airway (and mechanisms to deprive invadingmicroorganisms of ammonia) will be effective treatments for lunginfections, such as tuberculosis. A decline in airway pH would alsofavor protonation of the relatively stable NO-superoxide reactionproduct, peroxynitrite (OONO⁻) to peroxynitrous acid (OONOH),anoxidizing and nitrating species involved in macrophage-mediatedmycoplasma killing. These observations suggest that mild airwayacidification may be a subtle and titratable innate host defensemechanism which takes advantage of the pKa's of weak endogenous acids todefend the airway against airborne pathogens.

In accordance with one embodiment a method of treating microbialinfections of the airways comprises administering a composition thatresults in the acidification of the airways. In preferred embodimentsthe composition is delivered as an aerosol. In one embodiment thecomposition for lowering the pH of the airways comprises a proton donor,for example acetic acid, phosphoric acid, or dilute hydrochloric acid,or other proton donors. Alternatively, the composition may compriseS-nitrosothiols, such as S-nitrosoglutathione or S-Nitroso-N-acetylcysteine, not to kill the organism directly, but to inhibit glutaminase.In addition, the composition may comprise nebulizedDiazo-5-oxo-L-norleucine, an inhibitor of glutaminase. In accordancewith one embodiment a method for treating a Mycobacterium tuberculosisinfection comprises inhalation of a composition that lowers the pH ofthe airways to a range of about 6.0 to about 6.9.

EXAMPLE 1 Data Supporting the Hypothesis that Airway AcidificationContributes to the Pathophysiology of Acute Asthma

Eosinophil Necrosis and Release of Cytotoxic Mediators is Enhanced inAllergic Asthmatic Subjects by Endogenous Airway Acidification DuringViral Respiratory Infections.

Consistent with the established cellular toxicity of low pH-mediatedprotonation of NO_(x), applicants have recently described that humancosinophil necrosis (and overall cell death) are dramaticallyaccelerated in the upper end of the pH range observed during acuteasthma exacerbations compared to that observed in asymptomatic controls(Am J Respir Crit. Care Med 161:694-699; 2000). Further breathcondensate pH falls during HRV 16 infection. This drop in pH affectsboth the upper and lower airways. Direct measurement of nasal lavage pH(though diluted by saline) in high IgE asthmatic and control subjectsinfected with HRV 16 (n=3 each) revealed that the asthmatic subjects hadhigh ECP levels (258-668 ng/ml) associated with low pH (mean 6.9) whilecontrols had low ECP levels (8-77 ng/ml) associated with normal pH(7.5). These results are consistent with overall results showing thatHRV 16 infection causes an increase in nasal ECP levels selectively inthe high IgE-asthmatic group.

Experimental HRV 16 infection is associated with increased upper airwayECP levels. Ten non-allergic, non-asthmatic subjects, 11 subjects withasthma and relatively low IgE values (29.2-124 IU/mL) and six subjectswith high IgE values (371-820 IU/mL) and asthma were innoculatedintranasally with HRV 16 (500 tissue culture infective dose 50%(TCID₅₀)/mL, 2.5 mL in each nostril twice at time 0. Nasal lavage ECPlevels were measured sequentially for three weeks (See FIG. 4). Allgroups experienced an increase in upper airway ECP levels on the dayafter experiencing a fall in pH. Indeed, most subjects experience somedegree of fall in airway pH with inflammation. However, the rise in ECPwas substantially more pronounced in the high IgE-asthmatic group (p<)than in controls or lower-IgE asthmatics. This difference may bemultifactorial, but suggest that the allergic asthmatic response toairway acidification may involve increased release of inflammatorymediators.

The Cytotoxicity of Nitrogen Oxides Present in High Concentrations inthe Asthmatic Airway is Enhanced by a Fall in Lung Water pH.

Nitrogen oxide (NO_(x)) levels are elevated in the lower airway andbreath condensates of patients with asthma. It has been previouslyreported that asthma is associated with high breath condensate nitrite(NO₂ ⁻) and expired NO levels, consistent with evidence that nitricoxide synthase Type 2 (NOS 2) is chronically upregulated in theasthmatic lung. More recently, direct bronchoscopic evidence has beenobtained that nitrogen oxides (NO_(x)) (including the peroxynitrite(ONOO⁻) rearrangement product nitrate) tend to be elevated in the lowerairways of subjects with mild asthma (3±1 μM [n=8] vs. 1.7±0.3 μM incontrols [n=8]; p=0.06) and are increased in asthmatics relative tocontrols 48 hours after segmental antigen challenge (to 6±1 μM vs1.6±0.4 μM; p=0.02).

When exposed to acid, the airway NO_(x) present in high concentrationsin the baseline or antigen-challenged airway will be protonated, formingONOOH (pKa˜6.8) and HNO₂ (pKa˜3.4), compounds each substantially morecytotoxic than the corresponding base. Tissue injury caused by theprotonated species is mediated, at least in part, by disproportionatingreactions that form radical species including OH (from ONOOH), NO₂ (fromONOOH and HNO₂) and NO (from HNO₂) (Reactions 1 and 2). Consistent withdata from other groups suggesting accelerated ONOOH formation in theasthmatic airway, applicants have recently shown that reaction (1)proceeds rapidly during endogenous acidification of the asthmaticairway, consuming NO₂ ⁻ and producing NO (Am J Respir Crit. Care Med161:694-699; 2000). We have also shown that inhalation of an alkalineaerosol (0.05 mL/kg of 10 mM phosphate buffered saline [PBS]) in acuteasthma reverses this process, decreasing expired NO levels. Becausethere is now substantial evidence that expired NO values collectedaccording to ATS guidelines reflect lower airway (rather than nasal) NOevolution, this experiment also provides indirect evidence suggestingthat pH measurements made on breath condensate specimens reflect lowerairway chemistry.

EXAMPLE 2 Data Supporting the Hypothesis that Airway Acid is Buffered byAmmonia, a Product of Epithelial Glutaminase

Ammonium Concentrations are Low in Breath Condensate Specimens fromPatients with Acute Asthma.

Specimens were collected from patients admitted to the hospital or seenin the emergency department with acute exacerbations of asthma (low pH)or from healthy volunteers (no history of asthma or any otherobstructive lung disease and no chronic or acute illness). All patientswith asthma had acute expiratory flow limitation, with auscultatorywheezing, an inspiratory to expiratory time ratio of <½, and/or peakexpiratory flow or forced expiratory volume at one second (FEV₁) values<80% of predicted. The median NH₄ ⁺ concentration in asthmatic subjects(37; range, 0-442) was lower than in controls (314; range, 14-220) byMann-Whitney rank-sum testing (p<0.001). Logarithmically-transformed NH₄⁺ concentrations were correlated with logarithmically-transformed H⁺concentrations (pH) (r=0.59; p<0.001). Thus subjects with acute asthmaexacerbations who have a low breath condensate pH also have low breathcondensate NH₄ ⁺ levels (see FIG. 1).

Glutaminase Protein is Expressed in Human Airway Epithelial Cells.

Human kidney (KGA) and C-type (GAC) glutaminase transcripts are presentin human airway epithelial cells. Total RNA was isolated from confluentnormal human bronchial epithelial cells (NHBE; Clonetics) harvested bytrysinization. 10⁷ cell aliquots disrupted in lysis buffer (1%β-mercaptoethanol and guanidine isothiocyanate) and homogenized usingQIA shredder spin columns (Qiagen, Valencia, Calif.). Lysate was treatedwith 70% ethanol (1:1 v:v), applied to a RNeasy mini spin column(Qiagen), washed, eluted by centrifugation (10,000 g; 1 min) in 30 μldeioinized water and quantified by spectrophotometry. Aliquots of RNAwere mixed with KGA or GAC specific upstream and downstream or nesteddownstream primers and deoxynucleotides in RT-PCR buffer (ProStar HFsingle-tube RT-PCR system, Stratagene, La Jolla Calif.). Primers wereconstructed according to Elgadi, et al., (Physiol Genomics 1:51-62;1999). Moloney murine leukemia virus reverse transcriptase was added toeach reaction, with the exception of control reactions used to evaluatefor DNA contamination of the isolated RNA. In a control reaction,standard primers for the housekeeping gene glutaraldehyde phosphatedehydrogenase (GAPDH) were used. 0.5 μl of TaqPlus Precision DNApolymerase was added next. The reaction mixtures were placed in athermal cycler and subjected PCR amplification. The PCR products wereanalyzed by ethidium bromide stained 1% agarose gel electrophoresis incomparison to a 100 bp DNA ladder (GIBCO BRL, Grand Island, N.Y.). Thepredicted products were identified.

Glutaminase protein is expressed in human airway epithelial cells and isupregulated by corticosteroids in vivo. Open lung biopsy specimens froma subject with histologically normal lung who had been treated withsystemic corticosteroids for a month were compared with those from asubject with a severe asthma exacerbation who had been treated withsystemic corticosteroids for one day. Tissues were immunostainedsimultaneously and identically. Briefly, 3-5 μm formalin-fixed sectionswere deparaffinized and rehydrated with decreasing gradients of xyloseand of ethanol. Endogenous peroxidase activity was removed by incubationin 3% peroxide. Sections were blocked with normal serum and incubatedovernight (4° C.) with or without a polyclonal antibody to glutaminase(KGA). After washing, sections were incubated with or without (onesection each with only primary and only secondary antibody) goat-antirabbit biotinylated secondary antibody, followed by incubation with ABC(avidin-biotinylated horseradish peroxidase macromolecular complex)solution. Glutaminase staining was visualized using 3 3′diaminobenzidinesubstrate (Sigma). Sections were counterstained briefly withhematoxylin, dehydrated, and mounted using Permount. More glutaminaseimmunoreactivity (brown) in the airway epithelium of the control subjectwas observed than of the asthmatic subject and the same was true of thealveolar epithelium. The secondary antibody in the absence of primaryantibody (control) had no reactivity; and the same was true usingprimary antibody and no secondary antibody for both tissues.

Experimental HRV 16 Causes a Fall in Breath Condensate NH₄ ⁺Concentration AS Well as a Fall in Breath Condensate pH.

Consistent with observations made during acute asthma exacerbations,breath condensate NH₄ ⁺ concentrations fell from a median of 351 μM(range 151-1585) to 65 μM (range, 4-713; p<0.05) following experimentalHRV 16 innoculation. Additionally, breath condensate pH fell.

Th-1 Lymphocyte-Derived Cytokines Inhibit Airway Epithelial Glutaminase.

Acute infection with HRV may activate and/or recruited Th1 lymphocytesto the airway and may increase circulating and airway levels of IFN γ,which may worsen asthma symptoms. Th 1-derived IFN γ, in turn, is knownto inhibit glutaminase in human fibroblasts and in airway epithelialcells. Therefore, HRV 16 may acidify lung water in part through theeffect of IFN γ on glutaminase.

S—Nitrosothiols Inhibit Airway Epithelial Glutaminase.

S—Nitrosoglutathione (GSNO) is an endogenous neuroeffector and immunemodulatory molecule that is formed during NOS activation in neuronal andinflammatory cells. There is evidence that it is an, importantmodulatory compound in non-adrenergic, non-cholinergic autonomicsignaling. A proposed mechanism by which upper airway HRV infection maysignal lower airway symptoms in patients with asthma involves autonomicsignaling pathways, and GSNO is a signaling candidate.

GSNO has been demonstrated to inhibit airway epithelial glutaminaseactivity: Small airway epithelial cells. (SAEC: Clonetics) grown toconfluence in six well plates with 3 mM glutamine were incubated withS-Nitrosoglutathione (GSNO; 200 μm) in the presence or absence ofcysteinyl glycine (CG; 100 μM). Ammonium accumulation over time wascompared with control cells. S-Nitrosoglutathione require conversion toS-Nitrosocysteinyl glycine (CGSNO) (whether through simpletransnitrosation reactions in the presence of CG or through cleavage byγ glutamyl transpeptidase) to be fully active at intracellular sites,such as mitochondrial glutaminase, in some system. However, conversionof GSNO to CGSNO dramatically shortens the chemical and biologicalhalf-life of the SNO moiety. In this experiment, GSNO inhibited NH₄ ⁺accumulation at each time point (p<0.01 by ANOVA), an effect that wasaugmented at 24 hours by CO (p<0.01 compared with GSNO; no NH₄ ⁺ at allaccumulated). As expected, addition of CG eliminated the GSNO effectafter 48 hours because it accelerated catabolism. Accumulation of NH₄ ⁺in medium alone was at the threshold of detection. S-Nitrosothiols didnot affect cell viability as assessed by trypan blue/light microscopy.

Inhibition of airway epithelial glutaminase is likely to be the resultof post-translational interaction of the S-nitrosothiol with thecritical catalytic cysteine of glutaminase, consistent with similarmechanisms of regulation described for a large repertoire of otherproteins. Of note, it has been shown that NOS activation in airwayepithelial cells themselves may result from HRV-initiated IFN γsignaling, suggesting that S-nitrosylation may itself be the downstreambiochemical mechanism by which IFN γ inhibits glutaminase. Importantly,it has also been shown that activation of GSNO by γ glutamyltranspeptidase (γ GT) to S-Nitrosocyteinyl glycine is essential for manyof its bioactivities and also is involved in GSNO catabolism. Therefore,increased airway γ GT evident during acute asthma exacerbations couldalso account, at least in part, both for accelerated GSNO catabolism andfor inhibition of airway epithelial glutaminase.

EXAMPLE 3 Determining Airway Acidification Contributes to thePathophysiology of Acute Asthma Methods

Ammonium Measurement

Concentrations will be determined by the Sigma Diagnostics method.Briefly, condensed exhaled airway fluid vapor and supernatant fromnative tracheobronchial secretions will be reacted with NADPH in thepresence of L-glutamate dehydrogenase which reductively aminates added2-oxoglutarate. The decrease in absorbance at 340 nm reflects NADPHoxidation and is proportional to NH³ concentration.

Breath Condensate 3NT Measurements.

These will be performed as reported by Hanazawa and coworkers (Am JRespir Crit. Care Med 162:1273-1276, 2000). Subjects will breathethrough a mouthpiece and a two-way nonrebreathing valve which alsoserves as a saliva trap. Nitrotyrosine will be measured with a specificenzyme immunoassay according to the manufacturers instructions (CaymanChemical, Ann Arbor, Mich.).

Bronchial Epithelial Biopsy.

Following administration of topical lidocaine anesthesia, forceps willbe opened and advanced on the membranous side of the main, left andright carinae, closed and removed. Specimens will be placed initially insaline, and considered adequate if there is minor erythema and/orbleeding at the biopsy site, and if an inhomogeneous tissue sample atleast 1 mm in width is visualized in the saline. If the sample isinadequate, the site will be re-biopsied.

Bronchoscopic Airway pH Measurement.

Filter paper will be cut from Whatman 541 hardened ashless paper(Clifton, N.J.). Individual papers for the nasal studies were to 4 mm by1 cm, washed in double-distilled, deionized water and dried in an ovenovernight. pH neutralizing of the paper batch will be confirmed byplacing deionized water on the paper and reading the pH from a Cardymicro pH meter. Papers were folded in half width-wise, and weighed onthe day used (just before sampling). The end of the paper was grasped inthe forceps and positioned on the epithelial surface for 20 seconds. ThepH will be measured (Cardy) immediately after removing the forceps.Three measurements will be made on each subject.

Epithelial HRV 16 Infection.

Stocks of HRV 16 from Dr. Hayden's laboratory will be grown in confluentHeLa Ohio cells in minimal essential medium. Tissue culture infectivedose 50% (TCID₅₀) will be determined by viral titration in 96-wellplates of HeLa cells cultured for 5 days and evaluated for cytopathiceffect by fixation in methanol and staining with 0.1% crystal violet.Aliquots of HRV 16 will be inactivated with 1200 μJ/cm² uv light for 30min. Medium will be removed from epithelial cells grown to confluence in6-well plates and cells will be infected using 1 mL aliquots of activeor uv-inactivated HRV 16 for 8, 24, 48 and 72 hrs. Cells will be assayedfor glutaminase expression and activity at each time point.

Glutaminase Immunoprecipitation.

Cells will be lysed in 1 mL NP-40 buffer (150 mM NaCl, 1% NP-40, 50 mMTRIS, pH 8.0) containing protease inhibitors for 30 min (4° C.). TheLysate will undergo centrifugation (16,000 rpm; 10 min; 4° C.). Thelysate will be precleared of proteins which bind nonspecifically toantibody and protein A beads during a 2 hr incubation at 4° C. with 25mL protein A beads which have been preincubated with irrelevantantibody. The beads will be removed after a 5 second centrifugation andthe cleared supernatant will be incubated with 5 mg/250 mg proteinanti-rat kidney polyclonal-antibody (or 20 mg/mL IgG as control)overnight, then with 50 mL pre-quilibrated protein A beads for two hrs(each at 4° C.). Immunoprecipitates will be collected at 2,500 rpm (5min, 4° C.) and washed five times in 500 mM NaCl, 1% NP-40, 50 mM TRIS,pH 8.

Immunoperoxidase Staining.

This will be performed using VECTASTAIN Elite kit (Vector Laboratories)as described by the manufacturer. Briefly, serial sections offormalin-fixed paraffin-embedded lung will be mounted on precleanedSuperfrost Plus slides (Fisher Scientific). Tissue sections (3-5 μm)will be deparaffinize and rehydrated with a decreasing gradient ofethanol. Endogenous peroxidase activity will be removed by incubation in3% peroxide. Tissue sections will be blocked with normal serum andincubated overnight at 4-C with a polyclonal antibody for glutaminase(courteously supplied by Dr. Norman Curthoys). After washing to removeunbound antibody, sections will be incubated with goat-anti rabbitbiotinylated secondary antibody followed by incubation with ABC(avidin-biotinylated horseradish peroxidase macromolecular complex)solution. Controls will include reaction with primary antibody only,secondary antibody only and primary antibody pre-incubated with excessglutaminase. Glutaminase staining will be visualized using 3,3′diaminobenzidine substrate (Sigma). Sections will be counterstainedbriefly with hematoxylin, dehydrated, and mounted using Permount.

In Situ Hybridization (ISH).

This will be performed in lung tissue in order to identify the preciselocation and identification of cells containing glutaminase transcripts.In these studies, ISH will be performed on serial sections offormalin-fixed paraffin-embedded lung mounted on2-aminopropyltriethoxysilan coated slides. Two plasmids will beconstructed: (1) pBS480GAC which contains 480 bp of the GAC gene and (2)pBS640KGA which contains 640 bp of the KGA gene. Plasmids will belinearized and sense and antisense riboprobes transcribed using T7 or T3RNA polymerase. Transcripts will be subjected to limited alkalinehydrolysis to obtain a probe of approximately 150 nt. Initialexperiments will establish the optimal hybridization time andtemperature, washing conditions and exposure times. Riboprobes will betritium labeled to a specific activity of 1.1×10⁸ dpm/ml/kb. Sectionswill be autoradiographed, photographically developed, and counterstainedwith hematoxylin and eosin prior to microscopic observation usingbright-field and darkfield optics. RNA preservation of the specimenswill be assessed using a 1.8 kb probe directed against actin mRNA. Insitu hybridization data will be analyzed using both bright-fieldmorphology as well as darkfield optics to better visualize the fulldistribution of silver grains making up the autoradiographic signal.

Reverse Transcriptase PCR.

RNA will isolated from confluent cells (NHBE; Clonetics) harvested bytrypsinization. 10⁷ cell aliquots will be disrupted in lysis buffer (1%β-mercaptoethanol and guanidine isothiocyanate) and homogenized usingQIA shredder spin columns (Qiagen, Valencia, Calif.). Lysate will betreated with 70% ethanol (1:1 v:v), applied to a RNeasy mini spin column(Qiagen), washed, eluted by centrifugation (10,000 g; 1 min) in 30 μLdeioinized RNase-free water and quantified by spectrophotometry.Aliquots of RNA will be mixed with specific upstream and downstream ornested downstream primers and deoxynucleotides in RT-PCR buffer (ProStarHF single-tube RT-PCR system, Stratagene, La Jolla Calif.). Forrhinovirus (an RNA virus), isolation will be carried out as describedabove on specimens frozen immediately after collection (−70° C.) withaddition of 40 U RNase inhibitor (RNasin; Promega, Madison, Wis.)according to the method os Blomqvist, et al. Moloney murine leukemiavirus reverse transcriptase will be added to each reaction, with theexception of control reactions used to evaluate for DNA contamination ofthe isolated RNA. In a control lane, standard primers for thehousekeeping gene such as glutaraldehyde phosphate dehydrogenase (GAPDH)will be added. 0.5 μl of TaqPlus Precision DNA polymerase will then beadded and reaction mixtures placed in a thermal cycler and subjected tothe following program: 37 for 15 minutes to synthesize cDNA, 95° for 2minutes to deactivate the reverse transcriptase, and then 30 cycles of95° (30 sec), 58° (30 sec) and 68° (2 min), with a final 72° 10 minuteextension step. The PCR products will be analyzed by ethidium bromidestained 1% agarose gel electrophoresis in comparison to a 100 bp DNAladder (GIBCO BRL, Grand Island, N.Y.).

Airway Acidification is Associated with NO₂ ⁻ and ONOO⁻ Protonation InVivo.

As a first step, experiments will be designed to demonstrate that pH isa critical determinant of HNO₂ formation/airway NO evolution in subjectswith acute asthma exacerbation in vivo. It has been reported thatendogenously acidified breath condensate protonates NO₂ ⁻ ex vivo,decreases breath condensate NO₂ ⁻ concentrations, is associated withincreased expired NO concentrations and toxifies endogenous NO_(x) aspredicted, by reaction (1). This experiment extends those observationsto demonstrate that buffering endogenous airway acid inhibits reactionsin vivo. Further, this buffering experiment may also have directtherapeutic implications for patients experiencing acute asthmaexacerbations.

Phosphate buffer (10 mM) prepared in 10 mM normal saline (PBS) by theresearch pharmacist will be filtered (0.22 μm) and cultured to ensuresterility. Ten patients with acute asthma exacerbations (characterizedby a moderate increase in cough and wheeze, an FEV₁ of 40-70% predictedand a breath condensate pH<7.0) will be compared with 10 subjects withasthma who are not having an acute exacerbation (asymptomatic; FEV₁>70%predicted) and whose breath condensate pH is >7.0, as well as with 10healthy, control subjects (breath condensate >7.0). Subjects with acuteasthma exacerbations will be excluded if they are frankly dyspneic, havea respiratory rate >28 breaths/min, or have a room air oxygen saturationvalue <96%. Each subject will be given 0.05 mL/kg PBS by Pari-LC-plusnebulizer. Expired NO will be measured according to ATS standards (NIOX;Aerocrine AB; Sweden), before treatment and at 5, 10, 20, 30 and 40 minafter treatment.

Models for repeated measures (multiple time points) will be used to testthe hypothesis that expired NO over time differs among asthmaticsubjects having an acute asthma exacerbation, asthmatic subjects nothaving an acute exacerbation and healthy (control) subjects.Specifically, random coefficient models, which allow for subjectheterogeneity in NO profiles over time, will be used to compare thegroups. The time by group interaction is of primary interest as thisterm describes how the time course of expired NO differs among thegroups. Standard statistical software (SAS PROC MIXED) will be used tocarry out the analyses, including adjustments (Kenward-Roger smallsample corrections) to account for the relatively small sample sizes inthese experiments.

Effect of Experimental HRV Infection on Breath Condensate NO_(x).

A fall in pH associated with HRV 16 infection will result in NOprotonation and loss of relatively water-insoluble NO₂ ⁻ from solution(Reaction 1) as well as nitration of cellular proteins through ONOO⁻protonation. We will demonstrate that these cytotoxic reactions are morelikely to occur in asthmatic subjects than controls because of thegreater concentrations of NO_(x) salts in the asthmatic airway.

30 Adult subjects with (n=15) and without (n=15) asthma associated withIgE >200 u/mL (who are seronegative and culture/PCR negative for HRV 16)will be admitted to the general clinical research center (GCRC) andinoculated with 2.5 mL/nostril of 500 TCID-₅₀ units/mL of HRV 16.Subjects will be assessed daily for four days for twin infection bynasal washing for HRV 16 culture. Breath condensates will be collectedthrough a 0.3 μm filter (as well as using a simple saliva trap) atbaseline, then daily for four days, then weekly until three weeks afterthe initial inoculation. pH and NO₂ ⁻ levels will be measured on thefiltered condensates, as will amylase on the unfiltered specimens.Nitrated protein residues will be quantitated on the unfilteredspecimens according to the method of Hanazawa and reported per mgprotein. Random coefficient models will be used to compare dailyprofiles of nitrite (NO₂ ⁻) levels following HRV16 inoculation betweenhealthy controls and asthmatic subjects experiencing a fall in breathcondensate pH.

Intrathoracic Airway ECP and pH in Asthmatic Subjects Infected with HRV16 Who have Low Breath Condensate pH.

This experiment will provide 1) additional direct confirmation of therelevance of condensed expired air measurements to intrathoracic airwaypH; and 2) direct evidence that a fall in intrathoracic airway pHassociated with HRV 16 infection causes release of eosinophilicmediators uniquely in subjects with asthma. Intrathoracic airway pH willbe lower, and bronchoalveolar lavage (BAL) ECP levels will be higher, inthose high IgE-asthmatic subjects who have a fall in breath condensatepH 72 hours following HRV 16 inoculation than 1) they were before HRV 16challenge; and 2) in control subjects before and after HRV 16 challenge.

Subjects will be admitted to the GCRC and challenged with HRV 16according to the protocol described above. A subset of ten subjects withmild, high IgE (>200 u/mL) associated asthma and ten healthy controlswill be separately recruited from this group to undergo flexiblebronchoscopy before and 72 hours after HRV 16 inoculation. Eightsubjects in each group will be anticipated to complete this study. pHwill be measured using neutral, sterile filter paper introduced withforceps through the bronchoscope into trachea, the right and leftmainstem bronchi, allowed to become saturated with airway lining fluid,removed and assayed on a micro pH system. Bronchoalveolar lavage will beperformed in accordance with established guidelines and usingNO_(x)-free saline to lavage a subsegment of the right middle lobe(first bronchoscopy) and lingula (second). The fluid will undergocentrifugation (8,000 g; 5 min) and the supernatant will be frozen forECP measurement.

A one-sample t-test will be used to compare ECP levels pre- andpost-HRV16 inoculation among subjects experiencing a fall in breathcondensate pH. A two-sample t-test will be used to compare the pre- andpost-HRV16 changes to those observed in healthy controls. Based on ourpreliminary data regarding breath condensate pH and upper airway ECPlevels, eight high IgE-asthmatic subjects and 8 control subjects willgive us >90% power to detect differences in pH and ECP, when the truedifference is equal to the difference observed in the preliminary data.To correlate breath condensate to airway pH, mixed models will be usedto estimate the association and to account for the correlations inducedby having multiple pH samples per subject.

Relationship of Bronchoalveolar Lavage ECP Levels to BronchoalveolarLavage NO₂ and Nitrotyrosine Levels.

Levels of inert NO_(x) are elevated in the asthmatic airway at baseline.With acidification, these species become cytotoxic, causing eosinophilnecrosis in vitro. This experiment is designed directly to demonstrateThe relevance of these reactions to the mechanism by which HRV infectionand airway acidification leads to release of inflammatory mediators invivo. In BAL fluid of asthmatic patients experiencing an HRV 16infection, ECP levels are anticipated to be inversely related to [NO₂ ⁻]and directly related to [ONOO⁻]. Additionally, [NO₂ ⁻] and [ONOO⁻] inbreath condensates are anticipated to vary directly with [NO₂ ⁻] and[ONOO⁻], respectively, in BAL.

Lower airway pH will be measured, and BAL performed, following HRV 16infection as described above. Nitrite and 3-nitrotyrosine (3NT)/mgprotein (reflecting ONOO⁻ protonation) will be measured. Standardcorrelation and regression analyses will be used to measure theassociation between changes in ECP (dependent variable) levels and NO₂ ⁻and ONOO⁻ (independent variables) in asthmatic subjects experiencing afall in breath condensate pH.

EXAMPLE 4 Airway Acidification is Associated with Airway Narrowing inSubjects with Asthma, but not in Controls

Only patients with asthma will experience worsening expiratory airflowlimitation associated with a fall in airway pH. Because patients withasthma have a uniquely pH-sensitive airway environment, they willexperience increased airways inflammation and swelling followingHRV-induced acidification. This will be manifest physiologically asincreased air trapping caused by narrowing of the caliber of smallairways in patients with asthma, but not in controls. Measurement of RVand R_(aw) are very sensitive tests for expiratory flow limitation, andmay identify asthmatic changes even in the absence of a change inFEV₁/FVC. Therefore, increase in expiratory flow limitation will begreater in patients with high IgE asthma who have a fall in breathcondensate pH than 1) in patients with asthma who do not have a changein breath condensate pH; or 2) in control subjects following HRV 16infection.

Patients will be enrolled and studied as described in Example 1.FEV₁/forced vital capacity (FVC) ratio, residual volume (RV) and airwayresistance (R_(aw)) will be measured by spirometry and plethysmographyaccording to ATS guidelines. Physiological measurements will be madebefore HRV 16 infection and again at 72 hours following infection. Forpatients undergoing BAL, these measurements will be made before thebronchoscopy.

One-way analysis of variance will be used to test for changes in RV,FEV₁/FVC and R_(aw) among asthmatic subjects experiencing a fall in pH,asthmatic subjects not experiencing a fall in pH and control subjects.The specific comparisons of primary interest are comparing asthmaticsubjects with a fall in pH to subjects in each of the other two groups.Separate analyses will be done for each endpoint, with a Bonferronicorrection to the critical values to account for the multiple tests.

The Relationship Between Change in Breath Condensate pH and Degree ofObstruction.

We predict that breath condensate pH will be an important determinant ofairflow obstruction following HRV infection in patients with allergicasthma. As such, we predict that degree of change in acidity willpredict the degree of obstruction.

Residual volume to total lung capacity (TLC) ratio, FEV₁/FVC ratio,fraction of unventilated lung area in a single coronal mid-plane lungsection studied by single-breath hyperpolarized Helium MRI and breathcondensate pH will be studied before, and 72 hours following, infectionof patients with allergic asthma with HRV 16. Standard correlation andregression analyses will be used to estimate the association between pHlevels and degree of obstruction in subjects with allergic asthma. Theaddition of nonlinear terms to the model will be used to test theassumption of a linear relationship between obstruction and pH levels.

Relationship Between Lower Airway Acidification-Related Cytotoxicity andAir Trapping.

We anticipate that low airway pH will be associated with increasedcytotoxicity in the asthmatic airway, that neutralization of airway acidwill inhibit this cytotoxicity, and that low airway pH following HRV 16infection will be associated with the development of air trapping. Thus,we will complete the analysis of the pathway by demonstrating directlythat increased acidosis-related cytotoxicity is associated with airtrapping in allergic asthma.

Plethysmography and BAL will be performed before and after HRV 16 oneight subjects with high IgE asthma. Plethysmography will be performedbefore bronchoscopy. Change in RV/TLC will be studied as a function ofchange in ECP levels. Standard correlation and regression analyses willbe carried out to investigate the association between changes in RT/TLCand ECP levels.

EXAMPLE 5 Determination that Airway Acid is Buffered by NH₃, a Productof Epithelial Glutaminase

Acute Asthma Exacerbations and Airway Acidification are Associated witha Fall in NH₄ ⁺ Concentrations in Both Exhaled Breath Condensate andBronchoalveolar Lavage Samples.

This study will confirm our preliminary observations that low breath pHis accompanied by low breath NH₄ ⁺ levels in subjects with acute asthma,suggesting that impaired NH₄ ⁺ production may lead to acidification inthe airway, as it does in the renal tubular epithelium.

Twenty patients, four through 18 years old, presenting to the emergencydepartment between September and December who have active, β2agonist-reversible wheezing and meet NHLBI Expert Board guidelines for adiagnosis of asthma will be enrolled and asked to perform a breathcondensate collection as previously described by our group. Thesesubjects will be compared with a control group of wheeze-free childrenin the same age range who have no history of asthma. Patients will beexcluded from both groups if they have a history of hepatic, renal,metabolic, chronic inflammatory or acute infectious disease. pH valueswill be measured as previously described, and NH₄ ⁺ levels will bemeasured spectrophotometrically. Values of NH₄ ⁺ from subjects withasthma and low pH will be compared with those from control subjects.

Standard regression models will be used to estimate the associationbetween NH₄ ⁺ levels and pH levels in asthmatic and control patients.The first regression model will have NH₄ ⁺ levels as the dependentvariable and terms for group, pH level and a group by pH levelinteraction.

Airway Epithelial Cells Express Glutaminase that is Upregulated byCorticosteroids.

Human Airway Epithelial Cells Transcribe the Glutaminase Gene.

Transcription of genes for glutaminase by airway epithelial cells hasnot previously been demonstrated or studied. However, our preliminarydata demonstrate that glutaminase is transcribed and active in proximalhuman airway epithelial cells, and that its activity is relevant to theasthmatic response to viral infections. This experiment will demonstratetranscription of the gene in human primary cell culture lines from thedistal airway.

RNA will be extracted from A549 and SAEC cells grown to confluence insix-well plates as previously described. Primers constructed from thesequence for GAC and KGA isoforms of human glutaminase, as well asglyceraldehyde phosphate dehydrogenase (GAPDH; housekeeping) will beused to amplify a cDNA probe by reverse transcriptase PCR(RT-PCR). TheseRT-PCR products will be labeled with ³²P and used to probe mRNA fromnormal human bronchial epithelial (NHBE) cells and small airwayepithelial cells (SAEC) in Northern blot analysis. Glutaminase mRNA willbe identified by Northern blot and compared with that for GADPH bydensitometry. A two-sample t-test will be used to compare meanglutaminase mRNA by densitometry to GADPH.

Detection of Glutaminase RNA by In Situ Hybridization.

If glutaminase is an important determinant of HRV-initiated airway pHchanges and asthma exacerbations, it is likely to be downregulatedtranscriptionally by cytokines such as IFN γ (as is evident in othersystems and in vitro in the airway). This experiment will provide directin vivo evidence for this concept.

Patients undergoing bronchoscopy before and after HRV 16 infection willhave endobronchial epithelial biopsies obtained from the trachea andmainstem bronchi (n=3 biopsies from each at baseline and 72 hours afterinfection). Each biopsy will be studied for epithelial glutaminase mRNA(pro-mRNA can be used because of the post-transcriptionaldifferentiation of glutmaminase isoforms) by in situ hybridization.Specimens obtained from subjects whose pH has fallen to <7.0 will becompared to those from patients before HRV infection and to those whosepH has not fallen to <7.0. Repeated measures models for binary data willbe carried out, with one “between” factor (fall in breath pH group) andone “within” factor (biopsy location).

Effect of Hydrocortisone on Glutaminase Transcription

Glutaminase has a steroid-responsive promoter, and its expression isincreased by corticosteroids in a variety of systems. This biology fitswell with the observation that corticosteroids normalize airway pH invivo and improve asthma symptoms, as well as immunohistochemicalevidence for upregulation of human lung glutamine worse by systemiccorticosteroids. It is anticipated that glutaminase mRNA expression andactivity will be increased in airway epithelial cells in culturefollowing treatment with hydrocortisone.

mRNA will be extracted from airway epithelial cells after each cell typeis treated with hydrocortisone in a time (4, 6, 8, 12, and 24 hours) anddose (1, 10, and 100 μM-dependent fashion (four control wells and fourhydrocortisone wells per experiment) before harvest. Glutaminase mRNAwill be identified by northern blot, and compared with that for GAPDH bydensitometry. Activity will be compared at each point by NH₄ ⁺ assay in2 mM glutaminase. Two factor analysis of variance will be used toinvestigate the dependence of glutamine mRNA on time and dose ofhydrocortisone. Logistic regression models will be used to compare theprobability of expression as a function of time and dose ofhydrocortisone.

Effect of Airway Acid and of Interferon γ on Airway EpithelialGlutaminase Biochemical Activity.

In the renal tubular epithelium, glutaminase is upregulatedpost-transcriptionally by exposure to acidity in the glomerularfiltrate. We propose that airway epithelial glutaminase is similarlyupregulated by exposure to lumenal acid. Further, this effect of low pHon epithelial glutaminase activity should be overcome by IFN γ in vivo,consistent with observations that subjects developing acute viralinfection have low breath condensate NH₄ ⁺ levels in the context of lowpH. Therefore it is anticipated that 1) Exposure to a pH<7.4 (an airway“threshold” for acidification) will increase glutaminase activity ofhuman airway epithelial cells in culture; and that 2) Interferon γ willovercome this acidification effect and inhibit airway epithelial cellacidification at low and high pH.

Airway cells will be grown to confluence in six-well plates. One groupof cells will be exposed to pH 6.6 and another to pH 7.5, withpH-retitrated (HCl) every 12 hours, for 72 hours. Cumulative NH₄ ⁺production in the medium will be measured. Cell culture medium alone ateach pH level will serve as controls. The experiment will be repeatedwith 100 u/mL IFN γ in the culture medium—for the optimal timedetermined to see pH-induced glutaminase upregulation (above)—at low andhigh pH. A two-sample t-test will be used to compare the areas under thecurve between the group of cells exposed to pH 6.6 and the group exposedto pH 7.5.

EXAMPLE 6 Determining that Rhinovirus Infection Decreases AirwayEpithelial Glutaminase Activity

Glutaminase Activity and Expression During HRV 16-Associated LowerAirway Acidification In Vivo.

This experiment is designed to demonstrate directly the role ofglutaminase (and its level of regulation) in acidifying the airway inresponse to an HRV infection in vivo. We predict that HRV 16 infectionwill decrease airway epithelial glutaminase expression and activity invivo (even if it does not have a direct effect on epithelial cells invitro) because of T-ell and neuronally-mediated mechanisms in the intactorganism. It is anticipated that Bronchoalveolar NH₄ ⁺ levels and airwaybiopsy expression of glutaminase will be decreased in patients with HRV16 infection who have a fall in breath condensate pH.

Subjects will undergo breath condensate measurement, bronchoalveolarlavage and endobronchial biopsy before and after HRV 16 infection asdescribed in SA. Change in bronchoalveolar lavage NH₄ ⁺ concentrationand presence or absence of glutaminase expression will be characterizedas previously described, and compared before and after HRV 16 infectionsin patients who do and do not experience a fall in condensate pH. Thetwo-sample t-test will be used to compare changes in NH₄ ⁺ and thetwo-sample binomial test will be used to compare glutamine expressionbetween groups of subjects who experience a fall in breath condensate pHand those who do not experience a fall in breath condensate pH.

Interferon γ Inhibits Intrathoracic Airway Epithelial GlutaminaseExpression and Activity In Vitro and In Vivo.

Effect of IFN γ on Glutaminase Activity and Expression In Vitro

This experiment is designed to identify the optimal conditions at whichIFN γ, the primary candidate cytokine known to inhibit glutaminase inother systems, inhibits glutaminase in airway epithelial cells. Thedesign of this experiment will be similar to that of Example 2, exceptthat 1) all experiments will be done at pH 7.14, and 2) a dose response(1, 10, 100, and 500 u/mL) and time course (6, 12, 24, and 48 hr) forthe effect of IFN γ on all cells lines will be evaluated. We anticipatethat IFN γ will inhibit glutaminase in a dose- and time dependentfashion, and that this inhibition will be transcriptional.

In addition, condensed expired air pH and NH₄ ⁺ concentration are bothexpected to decrease following IFN γ therapy for idiopathic pulmonaryfibrosis (IPF). Bronchoalveolar lavage fluid will be obtained before andafter HRV 16 infection and analyzed for IFN γ concentration. Levels insubjects whose condensed expired air pH falls will be compared withthose from subjects whose pH does not change, and with values beforeinfection. We propose that Th1 (and/or CD 8) lymphocyte activation(whether in the airway or systemically) in response to HRV infectionwill increase BAL IFN γ levels. Also, a greater fall in subjects whoacidify will be consistent with the preceding experiments demonstratinga role for IFN γ in inhibiting glutaminase and regulating airway pH.

Effect of S-Nitrosoglutathione (GSNO) on Glutaminasc Activity in AirwayEpithelial Cells.

S-Nitrosylation reactions are increasingly appreciated to causefunctional post-translational modifications of proteins with criticalcysteine residues. This is particularly true of mitochondrial enzymessuch as glutaminase. S-Nitrosoglutathione is an endogenousS-nitrosylating agent that may be produced by nonadrenergic(noncholinergic) (NANC) neuronal stimulation and by inflammatory cells.As such, inhibition of glutaminase by S-nitrosylation reactions could bea mechanism by which an autonomic and/or inflammatory HRV-infected nasalmucosa to the lower airway mucosa to inhibit glutaminase and lower pH.

A549, NHBE and SAEC cells will be grown to confluence in 6 well platesin the presence of 2 mM glutamine and exposed to 0.5, 1, 10 and 100 μMGSNO for 4, 8 and 12 hours. Cumulative NH₄ ⁺ production will bedetermined calorimetrically. If there is inhibition of glutaminaseactivity, the effect of 100 μM dithiothreitol in reversing, and of 8bromo cyclic GMP in reproducing, this effect will be studied. Withineach cell line, two-factor analysis of variance methods will be used toinvestigate the effects of GSNO dose and time on activity. Similaranalyses will be conducted to investigate the roles of DTT and cGMP onactivity. We predict that lower airway epithelial glutaminase will beinhibited by GSNO in a time- and dose-dependent manner, that thisinhibition will be reversed by DTT and that it will not be reproduced by8-Br cGMP.

Role of γGT in the Inhibition of Glutaminase by GSNO.

It has been reported that γGT is critical for the bioactivation of GSNOin several models, including regulation of nuclear transcription factorsand control of breathing. Additionally, GSNO catabolism, mediated byγGT, may be accelerated in allergic asthma. We therefore anticipate thatγGT may be active in the airway epithelium, catabolizing GSNO andconverting it to cell-permeable, bioactive CGSNO, which then inhibitsglutaminase, allowing acidification of the airway. Defining this pathwaywill have important implications for developing new therapeutic agentsfor virally-induced asthma exacerbations. Accordingly, it is anticipatedthat acivicin will prevent the inhibition of airway epithelialglutaminase activity by GSNO, and this effect will be overcome by CGSNO.

Airway epithelial cells will be grown as described and treated with 100μM acivicin, an inhibitor of γGT. They will then be treated with GSNOand glutaminase activity will be measured. If acivicin blocks the effectof GSNO, we will examine the effect of acivicin in blocking theinhibition of glutaminase by 100 μM CGSNO. One-way ANOVA will be used tocompare the groups (GSNO, GSNO with acivicin) with respect toglutaminase activity

EXAMPLE 7 Practical Applications of the Present Invention

1. A 16 year old girl with moderate asthma develops an upper respiratoryinfection associated with wheezing and dyspnea. She receives treatmentwith albuterol. 0.083 mg diluted in normal saline (pH 3.5) but hersymptoms do not improve. She then receives another treatment with thesame dose of albuterol diluted in 10 mM phosphate buffered saline, pH 8,and her symptoms resolve.

2. A 45 year old man with severe asthma who becomes dyspneic whenever hedevelops an upper respiratory infection uses glutamine, 1 gm bynebulizer (pH 8) twice a day at the onset of upper respiratory symptoms.He does not develop an asthma exacerbation.

3. The same patient described in number 2 above develops an upperrespiratory infection the following winter. He takes glutamine, 1 gm bymouth three times a day and does not develop any asthma symptoms.

4. A 36 year old woman with severe asthma is unable to walk more than200 yards because of shortness of breath and stays up most nightscoughing. She is treated with levalbuterol and budesonide, 0.5 mg bynebulizer four times/day without relief of her symptoms. She beginsreceiving 1 mM glycine in 10 mM CAPS (pH 7.6) by nebulizer immediatelybefore her levalbuterol and budesonide treatments, and she becomesasymptomatic, with unlimited exercise tolerance and no cough at night.

5. A 50 year old woman with chronic yeast infection in her mouth hasworsening asthma. She is treated with a 7 day course of fluconazole 200mg/day and her asthma symptoms improve because of elimination of aceticacid-producing organisms.

6. The same woman described in number 5 above becomes recolonized with afermenting organism. Her asthma symptoms recur. She is treated with 10mg disulfiram by nebulizer 3 times/day to inhibit airway aldehydedehydrogenase activity. Her symptoms resolve.

7. A 5 year old boy with an upper respiratory infection-associatedasthma exacerbation is treated with 10 μM CuCl in 10 mM phosphatebuffered saline, pH 8, three times/day to break downglutaminase-inhibiting S-nitrosothiols. His asthma symptoms resolve.

8. A 40 year old woman with HIV is exposed to a friend with activepulmonary tuberculosis. She receives 1 mM diazo-5-oxo-L-norleucine (toinhibit glutaminase) in HCl (pH 2) by nebulizer three times daily for amonth and does not develop tuberculosis.

9. A 2 year old girl contracts active pulmonary tuberculosis caused byan organism resistant to conventional antimicrobial agents. She is begunon a low-glutamine diet and receives acetic acid (pH3) by nebulizer fourtimes daily for 9 months. Her symptoms resolve, and mycobacteria can nolonger be identified in her respiratory secretions.

1. A method for enhancing the therapeutic efficacy of one or moreagents, selected from the group consisting of corticosteroids,airway-smooth-muscle relaxants, and combinations thereof used in thetreatment of one or more airway diseases selected from the groupconsisting of asthma, asthma exacerbations, smooth muscle constriction,airway inflammation, overproduction of mucus, human rhinovirusinfection, common cold, allergic rhinitis, tuberculosis, cough, wheezeand airtrapping, said method comprising combining a non-toxic, alkalinecomposition with at least one said agent prior to bringing the resultingcomposition into contact with a patient's airways thereby alkalinizingsaid patient's airways.
 2. The method of claim 1 wherein saidcorticosteroids is selected from the group consisting of hydrocortisone,dexamethasone, and budesonide.
 3. The method of claim 1 wherein saidairway-smooth-muscle relaxants is selected from the group consisting ofalbuterol, levalbuterol, and ipratropium.
 4. The method of claim 1wherein said alkaline composition includes a basic buffer having analkaline pKa.
 5. The method of claim 4 wherein said buffer is selectedfrom one or more of the group consisting of acetate salts, ammoniumsalts, phosphate salts, bicarbonate salts, glutamine, glycine(aminoacetic acid), pK.sub.a 2=9.78), bicine(N,N-Bis(2-hydroxyethyl)glycine (pK.sub.a=8.46), tricene(N-[tris(hydroxymethyl)methyl] glycine (pK.sub.a=8.26), CAPS(3-(Cyclohexamino)-1-propanesulphonic acid (10.51), CAPSO(3-(Cyclohexamino)-2-hydroxypropanesulphonic acid (pK.sub.a 32 9.71),and 2-(Cyclohexamino)-ethenesulphonic acid (pKa=9.41).
 6. The method ofclaim 4 wherein said buffer is a natural or synthetic, non-toxic base.7. The method of claim 1 wherein said resulting composition is broughtinto contact with said patient's airways by inhalation.
 8. A methodcomprising: combining a non-toxic, alkaline composition with at leastone therapeutic agent selected from the group consisting ofcorticosteroids, airway-smooth-muscle relaxants, and combinationsthereof; and bringing the resulting combination into contact with apatient's airways thereby alkalinizing said patient's airways.
 9. Themethod of claim 8 wherein said at least one therapeutic agent is used inthe treatment of airway diseases selected from the group consisting ofasthma, asthma exacerbations, smooth muscle constriction, airwayinflammation, overproduction of mucus, human rhinovirus infection,common cold, allergic rhinitis, tuberculosis, cough, wheeze, andairtrapping.
 10. The method of claim 8 wherein said corticosteroids isselected from the group consisting of hydrocortisone, dexamethasone, andbudesonide.
 11. The method of claim 8 wherein said airway-smooth-musclerelaxants is selected from the group consisting of albuterol,levalbuterol, and ipratropium.
 12. The method of claim 8 wherein saidalkaline composition includes a basic buffer having an alkaline pKa. 13.The method of claim 12 wherein said buffer is selected from one or moreof the group consisting of acetate salts, ammonium salts, phosphatesalts, bicarbonate salts, glutamine, glycine (aminoacetic acid),pK.sub.a 2=9.78), bicine (N,N-Bis(2-hydroxyethyl)glycine(pK.sub.a=8.46), tricene (N-[tris(hydroxymethyl)methyl] glycine(pK.sub.a=8.26), CAPS (3-(Cyclohexamino)-1-propanesulphonic acid(10.51), CAPSO (3-(Cyclohexamino)-2-hydroxypropanesulphonic acid(pK.sub.a 32 9.71), and 2-(Cyclohexamino)-ethenesulphonic acid(pKa=9.41).
 14. The method of claim 12 wherein said buffer is a naturalor synthetic, non-toxic base.
 15. The method of claim 8 wherein saidresulting composition is brought into contact with said patient'sairways by inhalation.